Treatment of herniated intervertebral discs of mammals

ABSTRACT

Herniated intervertebral discs of mammals are treated to effect the selective in situ dissolution in vivo of nucleus pulposus and fibrocartilaginous tissue by the use of a sterile solution of purified collagenase injected into the intervertebral disc space.

O United States Patent [151 3,678,158 Sussman 51 July 18, 1972 [54]TREATMENT OF HERNIATED [56] References Cited INTERVERSTEBRAL DISCS OFUNITED STATES PATENTS Inventor: Bernard J. n, Washington, DC 3,320,1315/1967 Snnth ..424/94 [73] Assignee: Worthington BiochemicalCorporation, OTHER PUBLICATIONS Freehdd J. Clin. invest., 32 1323 1953[22] Filed: May 11, 1971 [21] APPL No; 142,333 Primary Examiner-AlbertT. Meyers Assistant Examiner-Frederick E. Waddell I Rel U- Applic ionDali! Attorney-Kenyon & Kenyon Reilly Carr & Chapin [63]Continuation-impart of Ser. No. 729,436, May 15,

1968, abandoned. ABSTRACT I Herniated intervenebral discs of mammals aretreated to ef- E (g! ..424/94 feet the selective in Sim dissolution invivo of nucleus p p 581 Field o'rsl'rc'iill'fffffffffffl'. ..424/94 andfibmcmilagimus tissue by the use of a Sterile Solution of purifiedcollagenase injected into the intervenebral disc space.

2 Claims, No Drawings TREATMENT OF HERNIATED INTERVERTEBRAL DISCS FMAMMALS This application is a continuation-in-part of my applicationSer. No. 729,436 filed May 15, 1968, now abandoned.

FIELD OF THE INVENTION This invention relates to the treatment ofherniated intervertebral discs of mammals and relates more especially totreatment whereby herniated intervertebral discs of mammals may besafely treated in vivo to relieve pressure on nerves and nerve rootswithout resort to surgical removal of the herniated disc.

DESCRIPTION OF THE PRIOR ART The pain associated with herniation of theintervertebral disc is related to compressive injury of the nerve rootoccasioned by dislocation of herniated tissue. Persistent symptomsappear to be related to further involvement of the root by chronicinflammation and edema. Rest and various forms of internal or externalimmobilization achieve their therapeutic effect by preventing furthertrauma to the root. Steroid therapy is employed for itsanti-inflammatory reaction and its reduction of edema. Foramenotomy anddisc removal are resorted to for the purpose of effecting nerve rootdecompression. More generally, those methods of treatment which aresuccessful allow the neural structure a greater amount of room. In orderto prevent recurrent pain and safeguard against multiple operativeprocedures, surgery is resorted to with increasing frequency as a majorintervention involving disc removal, foramenotomy and extensive bonefusion. However, such major therapy may fail to relieve symptoms.Moreover, it carries its own inherent risk and it may be unnecessarilydestructive of form and function.

Smith et al, Enzyme Dissolution of Nucleus Pulposus, Nature, 198, 1398(1963), using chymopapain, reported the dissolution of nucleus pulposusin rabbits. In this report and in subsequent papers (Enzyme Dissolutionof Nucleus Pulposus in Humans, J.A.M.A., 187, 137-140 1964) andTreatment of Lumbar Intervertebral Disc Lesions by Direct Injections ofChymopapain, J. of Bone & Joint Surg., Brit., 49-B, 502-519 1967) theauthors describe the'use of chymopapain in dogs with paraplegiasecondary to herniated disc and in human patients with sciatic syndrome.Favorable clinical results were claimed although complications occurredincludingone case of paraplegia and frequent instances of severe lowback pain. Widdowson, Effects of Chymopapain in the Intervertebral Discof the Dog, .I.A.V.M.A., 150, 608-617 (1967), working with chymopapainin dogs, confirmed the favorable effects in the disc space, butinjections into the spinal cord, subarachnoid or extradural spaces wereattended by hemorrhage, demyelination and satellitosis. In addition,this author reported that these complications, to a varying degree, wereconstant findings regardless of the site of injection. Shealy, TissueReactions to Chymopapain in Cats, J. of Neuro Surg., 26, 327-330 (1967),has further reported that chymopapain has no action specifically onnucleus pulposus and in fact causes severe necrosis wherever it isinjected, often producing death by subarachnoid and widespreadhemorrhage.

SUMMARY OF THE INVENTION It is an object of this invention to duplicatethe good results which are often obtained by minor operative proceduresby the selective dissolution of the herniated nucleus pulposus andannular fibrocartilage. It has been found, according to this invention,that this can be successfully accomplished essentially without injury toadjacent tissues, blood vessels or bone by the use of purified sterilecollagenase injected into the intervertebral disc space. As the resultof the injection, the nucleus pulposus and the fibrocartilaginous tissueof the disc and annulus, respectively, are selectively dissolved withresultant relief of pressure on the adjoining nerve root.

Collagenase is a rare enzyme found in certain clostridia culturefiltrates, and more especially culture filtrates of Clostridiumhirmlyticum and Clostridium welchii; the former being the preferredsource. As initially recovered, collagenase is impure and contains notonly collagenase but a peptidase and trypsinlike proteinase. Itsrecovery in impure form is described by Mandl et a1 (Isolation andCharacterization of Proteinase and Collagenase from C1. histolyticum, J.Clin. Invest, 32, 1323 (1953)). The use of this material has found someapplication in debridement of third degree burns, in enzymaticseparation of dermis and epidermis (Whole Mounts for the Study of Skinand its Appendages, .1. Invest. Dermatol., 23, 437-453 1954)).Collagenase also has been used in studies of different collagens byelectron microscopy (Evaluation of Structural and Chemical Changes inConnective Tissue, Annals NY. Acad. Sci., 56, 674-683 (1952)).Collagenase may be purified as by chromatography so as to recover anenzyme consisting essentially of collagenase. Moreover, by theemployment of conventional techniques to remove bacteria, thecollagenase may be produced in sterile form. Collagenase has beenpurified by electrophoresis of enzyme preparation obtained by ammoniumsulfate fractionation and has been the subject of biologicalinvestigation by Mandl et a1. (Clostridium Histolyticum Collagenase, ItsPurification and Properties, Archives of Biochem. and Biophysics, 74,465-475 (1958)) wherein its activity has been studied as regards itsability to attack collagen and its degradation products and itsinability to attack protein substrates such as casein or hemoglobin orthe fibrous proteins, fibrin, keratin and elastin.

Denatured collagen is susceptible to many proteolytic enzymes. However,unaltered collagen is resistant to all common proteolytic enzymes.Collagenase when used in accordance with this invention appears to be aunique microbial enzyme as regards its capacity to attack nativecollagen under physiological conditions of pH and temperature.

Collagen is found in various amounts in the body tissues such as theblood vessels, muscles, etc., and in the case of the material of thedisc and of the adjoining fibrocartilaginous material of the annulus ithas been found that by the use of a dilute solution of collagenase suchas a 0.1 percent solution injected into the disc space into contact withthe nucleus pulposus and annulus the collagenase solution will act onthe collagen content thereof with resultant substantially completesolution of the nucleus pulposus and solution of the major part of thefibrocartilaginous material of the annulus. The adjoining tissues suchas blood vessels and muscles also contain collagen; but in saidadjoining tissues the proportion of collagen relative to other tissuematerial that is not solubilized by collagenase is greater, and it hasbeen determined in practicing this invention that the enzyme exertslittle physiological or chemical effect on said adjoining tissues andthat there is virtually no damage to them as regards their integrity ortheir physiological functioning in the living mammal. Similarly, theenzyme does not have any apparent adverse effect on the adjoining bone.

For physiological usage in accordance with this invention, the enzymecan be recovered in the form of a purified stable solution thereof whichis subjected to lyophilization to produce it in dry powder form. The drypowder is stable almost indefinitely. At room temperature a 0.1 percentsolution is stable for 24 hours. Its stability is related to theconcentration of the enzyme, pH and the type of buffer which is usedwith it. Activity is optimal at a pH of 6.4 but there is a range ofactivity between 6.2 and 7.8. Moreover, its zone of maximum activity isaffected by the nature of any buffer that may be employed. When injectedinto the body of a mammal, the natural buffers of the body tend tomaintain a pH of about 7.4 and when, and even though, the pH of theenzyme may differ from this pH the body buffers tend to bring the pH ofan injected solution to a pH of approximately that of the body. Asaforesaid, the activity of collagenase is high at the pH and temperatureof the body. However, usually it is desirable to bring the pH of thesolution that is injected to a pH adjacent that of the body by the useof some buffer such as a phosphate buffer or the conventionalphysiological saline solution, namely, an

0.85 percent solution of sodium chloride which has a pH of about 7.While ordinarily the activity of the enzyme does not have to beinhibited after injection, nevertheless its activity can readily beinhibited by the employment of cysteine, low pH, 10 M p-chloromercuricbenzoic acid, IO M iodo acetic acid and various horse antisera.

In carrying out the invention, a purified and stable solution ofcollagenase, preferably about a 0.1 percent solution buffered toapproximately 7 pH, is injected directly into the disc space so as tocome into direct contact with the nucleus pulposus and surroundingannulus. The amount of solution depends on the size of the disc space.In the case of a medium sized dog, for example, approximately 0.5 to 1.0cc. usually is sufi'rcient, whereas in the case of a human the discspace is such size that the injection may be of the order of about 3 to5 cc. After the injection has been completed no further treatment isrequired other than such further observations as ordinarily are made inthe nature of X-ray examination, pulse, temperature, urinalysis, etc.

DETAILED DESCRIPTION AND EXAMPLES For the purpose of providing a betterunderstanding of this invention, it will be described hereinbelow inconnection with the following examples:

EXAMPLE 1 The method of this invention was utilized to effect selectivesolution of the nucleus pulposus and fibrocartilaginous material of thedisc and annulus in the spine of living dogs. This invention wassuccessfully demonstrated in connection with ten dogs in the followingmanner.

Each dog was first anaesthesized utilizing an intravenously administeredshort-acting barbiturate. incursion was effected through the lowerabdomen in nine cases and the paravertebral musculature in one case soas to afford operative retroperitoneal exposure and permit injectioninto the lumbar intervertebral disc space. In order to experimentallydetermine the safety of the treatment, the injection needle was advancedsufficiently so as to extend not only through the disc space but alsojust beyond and into the spinal canal but without penetrating the dura.2 cc. of a 0.1 percent aqueous solution of purified, sterile collagenasesolution were injected. Approximately 1 cc. of the solution remained inthe disc space. The remaining cc. of solution was discharged into thespinal canal although there was some leakage through the needle openingupon withdrawal of the needle. The treatment was effected in this way soas to fully test the absence of adverse side effects on adjacentmuscles, bone, blood vessels and more especially as regards possiblecontact with the dura. In ordinary practice the injection would becarried out so as to confine as much as possible of the injectedsolution in the disc space.

For a period of 7 to 10 days following the injection each of the dogsremained fully ambulatory. Blood count, urinalysis, temperature andpulse all remained within normal limits. Moreover, there were nodetectable neurological effects. After 7 to 10 days nine of the dogsand, after 2 days, one dog, were sacrificed and an X-ray of the treatedportion of the spine was compared with a corresponding X-ray made priorto the injection. Moreover, the affected section of the vertebral columnincluding adjoining vertebra was removed and cut in half so as to makethe affected region available for sectioning and photographic andmicroscopic study. In each case it was observed that the nucleuspulposus and a major portion of the fibrocartilaginous material of theannulus were completely absent and that there had been no significantattack on the hyaline cartilage, the anterior or posterior ligaments,the adjacent bone or the dura.

EXAMPLE 2 Two dogs were subjected to lumbar laminectomy wherein 2 cc. ofa sterile 0.1 percent solution of purified collagenase were injectedinto the extradural space. In the case of two additional dogs, 2 cc. ofthe same solution were injected intradurally. The two dogs whichreceived the injection extradurally were observed for 2 days. One of thedogs which received the injection intradurally was observed for 2 daysand the other for one week. In none of the dogs was there any evidenceof spinal cord injury. The dogs remained fully ambulatory with noweakness in the hind legs. There likewise was no evidence ofincontinence. The purpose of these tests was to demonstrate that evenunder extreme conditions of accidental injection extradurally or evenintradurally, the selective action of the purified collagenase is suchthat there is no apparent injury to the spinal cord.

EXAMPLE 3 During laminectomy on a living human patient material wasremoved from the disc space which responded to tests and microscopicexamination identifying it as nucleus pulposus or annulus fibrosis withsome admixture thereof. About 2 gram of the removed material consistingsubstantially entirely of nucleus pulposus was placed in 1 cc. of a 0.1percent solution of purified collagenase in sterile aqueous solution. Alike quantity consisting essentially of fibrocartilage was placed in acorresponding quantity of the 0.1 percent collagenase solution.Simultaneously a corresponding quantity of a small artery and of bone,respectively, was placed in a corresponding quantity of the 0.1 percentcollagenase solution in other vials. After 18 hours there wasessentially complete dissolution of the nucleus pulposus and of thefibrocartilage whereas there was no detectable effect on either theartery or the bone. This example demonstrates the capacity of thecollagenase to dissolve the nucleus pulposus and most of thefibrocartilage without detectable effect on adjacent blood vessels orbone.

EXAMPLE 4 Adjacent vertebra halves with a disc therebetween were removedfrom a human cadaver and subjected to dissection into component partsfor enzymatic attack on material in the disc space. Under theseconditions it is difficult to distinguish grossly between nucleuspulposus and degenerated fibrocartilage (annulus fibrosis).Nevertheless, in a separate test tube about is gram of nucleus pulposus,fibrocartilage, hyaline cartilage and anterior ligament, respectively,was immersed in 1 cc. of a 0.1 percent solution of purified collagenase.After 18 hours, dissolution of the nucleus pulposus and fibrocartilagewas complete. Under similar conditions of contact with the 0.1 percentsolution of collagenase the specimen of hyaline cartilage and thespecimen of anterior ligament were not appreciably attacked.

EXAMPLE 5 A disc specimen which had been obtained from a human cadaverand which was essentially similar to that described in example 4 wasdivided into five approximately equal parts. Each part was blotted withgauze and transferred to a sterile pre-weighed test tube. The tubes werereweighed thereby to obtain the weight of the sample and to each tubedifferent relative amounts were added of a 0.1 percent solution ofpurifled, sterile collagenase and of sterile 0.85 percent sodiumchloride so as to achieve a range of four different concentrations ofcollagenase while maintaining the volume of added solution constant at2.0 ml. The tubes were placed in a water bath at 37C. After seven hoursthe samples to which the collagenase solution had been added began tovisually reveal the beginning stages of solubilization. At the end of 24hours the solubilization observable as determined by visual inspectionwas substantial. The contents of each of the tubes was poured throughgauze and the residues were blotted with gauze to the TABLE 1 ml 0.1% mllnitial Final Colla 0.85%Colla- Weight Weight 96 Solu- Sample genaseNaCl genase gm.) (gm.) bilized a 0.2 1.8 0.010 1.137 0.938 17.5 b 0.51.5 0.025 1.028 0.636 38.2 c 1.0 1.0 0.050 0.692 0.323 58.3 d 2.0 0.1000.769 0.205 73.4 e 0 2.0 0 0.933 1.046

The data of the foregoing table show that while the percentsolubilization of the material of an unhemiated disc obtained from acadaver progressively decreases at decreasing collagenase concentrationsless than 0.1 percent, it is noteworthy that even at 0.01 percentconcentration the solubilization obtained is substantial.

EXAMPLE 6 A specimen of an herniated intervertebral disc removed from aliving human during surgery was rinsed aseptically twice with sterile0.85 percent sodium chloride to remove excess blood. The specimen wasdivided into five approximately equal parts which were treatedidentically according to the procedure described in example to provide arange of four concentrations of collagenase while maintaining the volumeof added solution constant at 2.0 ml. The tubes were placed in a waterbath at 37C. Solubilization was apparent after 2 or 3 hours and was verypronounced after seven hours. After 24 hours the contents of each tubewas poured through gauze in an attempt to weigh the residue, as had beendone in example 5. However, in each of the tubes containing enzymesolution the solubilization had progressed so far that any slightresidue was not palpable and was impossible to weigh. Virtually all ofthe specimen was solubilized in each of the tubes containing enzymesolution. The data for the test samples and the test results are setforth in the following table:

The increase in solubillzation as compared with example 5 reflects thegreater collagenase content of the herniated disc used in example 6. Thetest data show that in the treatment of an herniated disc highlyeffective solubilization of the disc substance was obtained whenemploying a solution of the purifled collagenase at concentrations ofapproximately 0.01 percent to approximately 0.1 percent.

The practice of this invention is of utility in connection withveterinarian usage as well as in humans. For example, dogs, particularlyof the dachshund breed, are subject to injury with resultant herniationof spinal discs. 1n the treatment of humans it is contemplated to effectentry into the disc space for injection of the collagenase solution fromthe back of the patient with the injection needle passing through thedura and spinal fluid, and through the posterior ligament using atechnique similar to that which is conventional to a spinal tap for theremoval of spinal fluid except that introduction of the needle iscarried further into the disc space. Alternatively, so that the needlewill not come into proximity with the dura a lateral approach to thedisc space may be employed. Whether the collagenase is used forveterinarian purposes or for the treatment of humans, conventional X-raytechniques may be employed for locating the herniated disc.

It is a further aspect of this invention that collagenase is provided indosage form appropriate for a single intervertebral injection into aliving mammal and, to this end, a sterile, purified solution ofcollagenase is introduced into a vial in sufiicient quantity to providethe necessary volume for injection of a solution having a concentrationof approximately 0.01 to 0.1 percent. The solution is then subjected tolyophilization to produce the collagenase in dry, sterile condition andthe vial is sealed. At the time of use the aqueous medium used forinjection is added directly to the vial. As aforesaid, the aqueous solvent may contain a small amount of buffer or saline material to providethe desired pH. The collagenase solution is ready for use as soon as itis prepared in this way.

Known procedures for the purification of the collagenase may be employedsuch as the procedure described by Keller and Mandl (The Preparation ofPurified Collagenase, Arch. of Biochem. and Biophys, 101, 81 (1963)). Asa precautionary measure, a test portion of the collagenase that isproduced for clinical use should be tested to insure its essentialfreedom from proteolytic and elastolytic activity. Desirably also itshould be checked for its sterility and pyrogenicity. The preferredminimum activity for collagenase is 200 units which may be determined bythe assay method of Mandl et al. (lsolation and Characterization ofProteinase and Collagenase from Cl. histolyticum, J. Clin. Invest., 32,1323 (1953)). The amino acids liberated are expressed as micromolesleucine per milligram collagenase.

1 claim:

1. In the treatment of an herniated intervertebral disc wherein there isinjected into the disc space a solution of an enzyme which selectivelydissolves material comprised in the disc, the improvement which consistsessentially of the injection into the disc space of a sterilepharmaceutically acceptable aqueous solution of purified collagenase theconcentration of which is from approximately 0.01 percent toapproximately 0.1 percent in an amount that is effective to selectivelydissolve native collagen contained in the nucleus pulposus and in theadjoining annulus fibrosis, said collagenase being recovered fromclostridia culture filtrates selected from the group consisting ofClostridium histolyticum and Clostridium welchii and being essentiallyfree of proteolytic activity on any protein other than collagen.

2. The method of claim 1 wherein the concentration of the collagenase inthe injected solution is approximately 0.1 percent.

2. The method of claim 1 wherein the concentration of the collagenase inthe injected solution is approximately 0.1 percent.